Ubiquitin-Specific Proteases (UBPs) are a conserved family of proteins in eukaryotes that play critical roles in protein de-ubiquitination. The covalent modification of proteins by ubiquitin play a central role in diverse cellular pathways such as cell cycle progression, signal transduction, transcriptional regulation, DNA repair, stress responses, endocytosis and apoptosis (Hochstrasser, 1996; Varshaysky, 1997; Hershko and Ciechanover, 1998; Weissman, 2001; Pickart, 2004). Protein ubiquitination is catalyzed by a cascade of three enzymes. Ubiquitn is first activated by ubiquitin-activating enzyme (E1), which forms a thiolester bond with the ubiquitin C-terminus. Ubiquitin is then transferred to ubiquitin-conjugating enzyme (E2). Although some E2s can catalyze ligation of the ubiquitin C-terminus to the lysine residue of target proteins with the aid of ubiquitin ligases (E3s), other E2s transfer their conjugated ubiquitin to E3s before targetting to substrates. Target substrate proteins can be mono-ubiquitinated or multi-ubiquitinated by successive conjugation of the ubiquitin C-terminus to the lysine residue of the prior one through several possible linkages. The fate of ubiquitinated substrate proteins depends in part on the number of conjugated ubiquitin(s) and on the mode of linkage in the ubiquitin chain. The most common ubiquitination is multi-ubiquitin chain (ubiquitin number>=4) linked by Lys48, acting as a signal for protein degradation by 26S proteasome.
Cleavage of ubiquitin from proteins by de-ubiquitination enzymes (DUBs) can also affect the ubiquitinated substrate protein's activity and fate (Wilkinson, 1997; Amerik and Hochstrasser, 2004; Crosas et al., 2006; Hanna et al., 2006). Those DUBs are proteases that specifically cleave the peptide bond between ubiquitins or between the C-terminus of ubiquitin and covalently attached polypeptides. The currently known DUBs together carry out four types of essential biochemical functions: first, they generate mature ubiquitins from ubiquitin precursors (fused to ribosomal protein) and polyubiquitin gene products; secondly, they rescue proteins that are inappropriately ubiquitinated; thirdly, they cleave ubiquitin (chains) from attached substrate proteins; and fourth, release free ubiquitin monomers from multi-ubiquitin chains. The last three roles are accompanied by cleavage of the isopeptide bonds between the ubiquitin C-terminus Gly and Lys ε-amino residue of a target protein.
Cysteine proteases and metalloproteases are the two major groups of the DUB superfamily, with cysteine proteases being most numerous in eukaryotes (Nijman et al., 2005). All known metalloproteases have a JAMM domain for catalytic activity (Verma et al., 2002). The cysteine protease DUBs can be further divided into four families based on the organization of ubiquitin-protease catalytic center structure and organization (Wilkinson, 1997; Amerik and Hochstrasser, 2004; Nijman et al., 2005). Ubiquitin-Specific Proteases (UBP, or USPs as defined in mammals) possess catalytic triad residues in highly conserved cysteine box and histidine box (Hu et al., 2002). Ubiquitin C-terminal Hydrolases (UCHs), with similar catalytic triad residues in two conserved cysteine and histidine boxes (Johnston et al., 1997; Johnston et al., 1999), have a smaller overall protein size as well as a structural obstacle over the catalytic surface to restrict their ability to hydrolyze only small amides and esters at the C-terminus of ubiquitin (Amerik and Hochstrasser, 2004). Ovarian Tumor Proteases (OTUs) have a catalytic triad comparable to above two families in Cysteine and Histidine boxes but containing an OTU-related motif and being considered as a part of UBP family (Balakirev et al., 2003; Nanao et al., 2004). Lastly, Machado-Joseph Disease Protein Domain Proteases (MJDs) possess a cysteine and histidine box-like domain but with rather low sequence similarity to the other three groups (Burnett et al., 2003; Scheel et al., 2003). The UBP family makes up the bulk of the cysteine proteases. All four types of the above mentioned DUB biochemical functions are found in UBPs family, while UCHs only perform their functions on small proteins and ubiquitin precursor.
In the model plant Arabidopsis thaliana, an in silico analysis of the completely sequenced genome revealed a total of 27 UBPs based on the presence of the conserved Cys and His boxes; those 27 UBPs were further divided into 14 subfamilies (Yan et al., 2000). Previous reports showed that UBP3 and UBP4 constitute one subfamily, possess UBP activity in vitro and are present in the nucleus (Chandler et al., 1997; Rao-Naik et al., 2000). Another member, UBP5, was shown also to have de-ubiquitination activity in vitro (Rao-Naik et al., 2000). A genetic analysis of UBP1 and UBP2, members of another subfamily, were reported to be required for resistance to the amino acid analog canavanine (Yan et al., 2000). Furthermore, a loss-of-function mutation in UBP14 was shown to be lethal in early embryo development (Doelling et al., 2001).